Silver stained polyacrylamide gel for DNA mutation analysis too dark

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che.chattha
che.chattha's picture
Silver stained polyacrylamide gel for DNA mutation analysis too dark

We are setting up a diagnostic lab protocol for prenatal diagnosis and mutation analysis of Beta Thalassaemia major. We extract the DNA from whole blood samples using Chellex extraction method and then amplify the product by PCR. The problem we are facing is at the gel staining stage. We use a 6% polyacrylamide gel in TBE Buffer at 200V for 30min. Then the gel is stained by first taking out and putting in 0.1% AgNO3 (silver nitrate) for 20 min, then washing for  2 times using distilled water and then soaking the gel in 1.5% Sodium Hydroxide with 200ul in 100ml formaline untill bands develop and then transfered to filter paper to preserve.

The problem is that the gel comes out too dark (as in black and almost unreadable) as soon as it is put in the dryer to dry.
the gel dryer conditions that we use are 70 degree celcius for 30min at 30 bars. the thing is that the gel looks fine right after stainig but as soon as we put it in the dryer it starts to darken quickly (within 45 seconds).

please help me figure out the problem. Thanks.

Kristina Holmberg
Kristina Holmberg's picture
 Based on your protocol, it

 Based on your protocol, it looks like you are not stopping the development using e.g. 10% acetic acid. I have attached a PDF with an article comparing different silver staining methods that might be helpful.